Method for production of hepatic-lobule-like cell cluster from adipose-tissue-derived cell

ABSTRACT

Disclosed are: a method for producing a hepatic-lobule-like cell mass from an adipose-tissue-derived cell, which is characterized by culturing the adipose-tissue-derived cell; a hepatic-lobule-like cell mass produced by the method; a method for the screening of a substance capable of promoting or inhibiting the formation of a hepatic-lobule-like cell mass, which is characterized by culturing an adipose-tissue-derived cell to produce the hepatic-lobule-like cell mass, wherein a candidate substance is added to a culture medium; and a kit for use in the screening method.

TECHNICAL FIELD

The present invention relates to a method for obtaining a hepatic lobulecell population from an adipose-tissue-derived cell, a hepatic lobulecell population obtainable thereby, a method of screening for asubstance that promotes or inhibits formation of hepatic lobule and fora substance that causes the activity of a hepatic lobule to increase ordecrease, and a kit therefor, and the like.

BACKGROUND ART

Compared to other countries, Japan has a high incidence of hepatitis C.That chronic hepatitis, hepatic cirrhosis and then liver cancer occurdue to infection by hepatitis C virus is a known fact. Medical therapiesmainly centered on interferon are considered to be effective in chronichepatitis and hepatic cirrhosis states. Meanwhile, once liver canceroccurs, a complete cure is medically impossible, leaving surgicalextraction of the tumor as the number one choice. However, in reality,since advanced hepatic cirrhosis is to be the stage where liver cancerwill occur, only surgical treatments can be selected also for liverfailure, and even therapies such as TAE or PEIT cannot be attempted ifthe case becomes serious. Liver transplantation has been performed onsuch serious hepatic cirrhosis patients for a long time. However,regarding cerebrally dead donor liver transplantation, the absoluteinsufficiency of donors, and regarding living donor livertransplantation, ethical issues such as safety for the donor, andinsertion of a surgical knife into a healthy subject exist. Thus, theidea of a treatment of liver failure by hepatocyte regeneration wasborn.

In recent years, the presence in adipose tissues of a cell populationthat trans-differentiates and may differentiate into cerebral nervous,musculoskeletal, cardiovascular and endocrine constituent cells,similarly to bone-marrow-derived mesenchymal stem cells and cordblood-derived stem cells, was reported (Non-patent Reference 1). Sincecollection of adipose tissue is simple, only little ethical issues areconsidered to exist if the collected adipose-tissue-derived mesenchymalstem cell were used in not only the subject it is derived from, but evenin a patient other than such subject.

Regarding the liver, a report exists, in which a subcutaneoustissue-derived stem cell was used for causing differentiation intohepatocyte (Non-patent Reference 2). However, there was no report that ahepatic lobule could be regenerated, which is the smallest functionalunit of the liver, that can be used in a treatment.

[Non-patent Reference 1]

-   Gmble J. et al., Cytotherapy. 2003; 5(5): 362-9

[Non-patent Reference 2]

-   Seo M J. et al., Biochem Biophys Res Commun. 2005 Mar. 4; 328(1):    258-64

An object of the present invention is to provide a method for causing ahepatic lobule cell population, which is the smallest functional unit ofthe liver, to be formed from an adipose-tissue-derived cell, and ahepatic lobule cell population obtainable thereby. In addition, it is anobject of the present invention to develop methods for screening for asubstance that promotes or inhibits formation of hepatic lobule and fora substance that causes the activity of a hepatic lobule to elevate ordecrease, and kits therefor.

In view of the above situation, the present inventors conducted earneststudies and discovered as a result that a hepatic-lobule-like cellcluster may be caused to form with a high probability by culturing, andin particular by culturing in a suspended state, an undifferentiatedcell obtained from adipose-tissue-derived cells, and reached completionof the present invention.

DISCLOSURE OF THE INVENTION

That is to say, the present invention provides

(1) a method for obtaining a hepatic-lobule-like cell cluster from anadipose-tissue-derived cell, the method comprising: culturing anadipose-tissue-derived cell,

(2) the method described in (1), comprising the step of obtaining ahepatic-lobule-like cell cluster from an undifferentiated cell,

(3) the method described in (1) or (2), comprising the steps of:

(a) obtaining an undifferentiated cell from an adipose-tissue-derivedcell, and

(b) obtaining a hepatic-lobule-like cell cluster from theundifferentiated cell,

(4) the method described in (2) or (3), in which the step of obtaining ahepatic-lobule-like cell cluster from an undifferentiated cell is a stepof producing a hepatic-lobule-like cell cluster by culturing anundifferentiated cell in a suspended state,

(5) a hepatic-lobule-like cell cluster obtainable by the methoddescribed in any of (1) to (4),

(6) a hepatocyte contained in the hepatic-lobule-like cell clusterdescribed in (5),

(7) a medicinal composition for preventing or treating a disease thatoccurs due to deterioration in a liver function, the compositioncontaining the hepatic-lobule-like cell cluster described in (5) and/orthe hepatocyte described in (6),

(8) use of the hepatic-lobule-like cell cluster described in (5) and/orthe hepatocyte described in (6) for production of a medicinal drug forpreventing or treating a disease that occurs due to deterioration in aliver function,

(9) a method for treating or preventing a disease that occurs due todeterioration in a liver function, said method comprising: administeringthe hepatic-lobule-like cell cluster described in (5) and/or thehepatocyte described in (6),

(10) a method of screening for a substance that promotes formation ofhepatic lobule, said method comprising: adding a candidate substance toa culture medium when culturing an adipose-tissue-derived cell to obtaina hepatic-lobule-like cell cluster, wherein the candidate substance is asubstance that promotes formation of hepatic lobule when formation ofhepatic-lobule-like cell cluster is promoted in comparison withformation in a system not containing the candidate substance,

(11) a substance obtainable by the method described in (10), whichpromotes formation of hepatic lobule,

(12) a method of screening for a substance that inhibits formation ofhepatic lobule, said method comprising: adding a candidate substance toa culture medium when culturing an adipose-tissue-derived cell to obtaina hepatic-lobule-like cell cluster, wherein the candidate substance is asubstance that inhibits formation of a hepatic lobule when formation ofhepatic-lobule-like cell cluster is inhibited in comparison withformation in a system not containing the candidate substance,

(13) a substance obtainable by the method described in (12), whichinhibits formation of hepatic lobule,

(14) a kit for screening for a substance that promotes or inhibitsformation of hepatic lobule to be used in the method described in (10)or (12),

(15) a method of screening for a substance that causes an activity of ahepatic lobule to increase, said method comprising: culturing, in aculture medium containing a candidate substance, a hepatic-lobule-likecell cluster that has been obtained by culturing anadipose-tissue-derived cell, wherein the candidate substance is asubstance that causes the activity of a hepatic lobule to increase whenan activity of the hepatic-lobule-like cell cluster has increased incomparison with an activity in a system not containing the candidatesubstance,

(16) a substance obtainable by the method described in (15), whichcauses an activity of a hepatic lobule to increase,

(17) a method of screening for a substance that causes the activity of ahepatic lobule to decrease, said method comprising: culturing, in aculture medium containing a candidate substance, a hepatic-lobule-likecell cluster that has been obtained by culturing anadipose-tissue-derived cell, wherein the candidate substance is asubstance that causes an activity of a hepatic lobule to decrease whenthe activity of the hepatic-lobule-like cell cluster has decreased incomparison with an activity in a system not containing the candidatesubstance,

(18) a substance obtainable by the method described in (17), whichcauses the activity of a hepatic lobule to decrease, and

(19) a kit for screening for a substance which causes an activity of ahepatic lobule to increase or decrease, said kit being used in themethod according to (15) or (17).

According to the present invention, a method for obtaining ahepatic-lobule-like cell cluster from an adipose-tissue-derived cell,and a hepatic-lobule-like cell cluster obtainable thereby, a method ofscreening for a substance that promotes or inhibits formation of hepaticlobule and a substance obtainable thereby which promotes or inhibitsformation of hepatic lobule, a method of screening for a substance thatcauses the activity of a hepatic lobule to increase or decrease, and asubstance obtainable thereby which causes the activity of a hepaticlobule to increase or decrease, as well as kits for such screenings, andthe like, are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a micrograph of the obtained adipose tissue-derived cells.

FIG. 2 is a micrograph of the obtained adipospheres.

FIG. 3 is a micrograph of the obtained hepatic-lobule-like cell cluster.

FIG. 4 is a graph showing the results of quantitative RT-PCR forα-fetoprotein. The vertical axis represents the ratio of the expressionof α-fetoprotein with respect to the expression of GAPDH.

FIG. 5 is a graph showing the results of quantitative RT-PCR foralbumin. The vertical axis represents the ratio of the expression ofalbumin with respect to the expression of GAPDH.

FIG. 6 is a graph showing the results of quantitative RT-PCR for keratin18. The vertical axis represents the ratio of the expression of keratin18 with respect to the expression of GAPDH.

FIG. 7 is a graph showing the results of quantitative RT-PCR for keratin19. The vertical axis represents the ratio of the expression of keratin19 with respect to the expression of GAPDH.

FIG. 8 is a graph showing the results of quantitative RT-PCR for CYP1B1.The vertical axis represents the ratio of the expression of CYP1B1 withrespect to the expression of GAPDH.

FIG. 9 is a graph showing the results of quantitative RT-PCR forglutamine synthase. The vertical axis represents the ratio of theexpression of glutamine synthase with respect to the expression ofGAPDH.

FIG. 10 is the result of western blot analysis for the α-fetoproteinproduced by the hepatic-lobule-like cell cluster.

FIG. 11 is the result of western blot analysis for albumin produced bythe hepatic-lobule-like cell cluster.

FIG. 12 is a micrograph by immunohistochemical staining showing thepresence of α-fetoprotein in a hepatic-lobule-like cell cluster.

FIG. 13 is a micrograph by immunohistochemical staining showing thepresence of albumin in a hepatic-lobule-like cell cluster.

FIG. 14 is a fluorescence micrograph showing the incorporation ofDiI-LDL in a hepatic lobule cell population.

FIG. 15 is a micrograph showing the accumulation of glycogen in ahepatic lobule cell population by PAS staining.

FIG. 16 is a graph showing the amount of urea generated by ahepatic-lobule-like cell cluster.

FIG. 17 is a graph showing the effects from the grafting of ahepatic-lobule-like cell cluster in a hepatitis mouse model. Thevertical axis represents total bilirubin concentration (mg/dL).

FIG. 18 micrograph by HE staining showing a survival ofhepatic-lobule-like cell cluster after grafting of thehepatic-lobule-like cell cluster under the renal capsule[2] of mouse.

FIG. 19 is a micrograph by immunohistological staining showing theexpression of albumin in a hepatic-lobule-like cell cluster aftergrafting.

FIG. 20 is a micrograph by PAS staining showing the accumulation ofglycogen in a hepatic-lobule-like cell cluster after grafting.

FIG. 21 is a micrograph of ADMPC obtained from an adipose tissue.

FIG. 22 shows the expression of a neural crest[1] cell-specific markergene from the results of RT-PCR using ADMPC-derived RNA.

FIG. 23 is an electrophoretic image showing the results of RT-PCR usingADMPC-derived RNA.

FIG. 24 shows the results of RT-PCR when ADMPC was sub-cultured sixtimes.

FIG. 25 is a graph showing the amount of expression by quantitative PCRof Sca-1 in ADMPC. The vertical axis represents Sca-1/GAPDH.

FIG. 26 is a graph showing the amount of expression by quantitative PCRof ABCG2 in ADMPC. The vertical axis represents ABCG2/GAPDH.

FIG. 27 is the result of FACS analysis showing the expression of SSEA-4,CD29, CD44, CD73, CD105 and CD166 in ADMPC.

FIG. 28 is the result of FACS analysis showing that fibroblasticcontamination is small in ADMPC compared to ADSC from a conventionalmethod.

FIG. 29 is a micrograph of pancreatic endocrine cells obtained bycausing ADMPC to differentiate.

FIG. 30 is a micrograph of hepatocytes obtained by causing ADMPC todifferentiate.

FIG. 31 is a graph showing the amount of expression by quantitative PCRof α-fetoprotein in hepatocytes obtained by causing ADMPC todifferentiate. The vertical axis represents AFP (α-fetoprotein)/GAPDH.

FIG. 32 is a graph showing the amount of expression by quantitative PCRof albumin in hepatocytes obtained by causing ADMPC to differentiate.The vertical axis represents albumin/GAPDH.

FIG. 33 is a graph showing the amount of expression by quantitative PCRof CYP1B1 in hepatocytes obtained by causing ADMPC to differentiate. Thevertical axis represents CYP1B1/GAPDH.

FIG. 34 is a graph showing the amount of expression by quantitative PCRof glutamine synthase in hepatocytes obtained by causing ADMPC todifferentiate. The vertical axis represents glutamine synthase/GAPDH.

FIG. 35 shows the results of RT-PCR in cells cultured in the presence ofDMSO or OP9 culture supernatant.

FIG. 36 is a graph showing the expression by quantitative PCR of Nk×2.5in cells cultured in the presence of DMSO. The vertical axis representsNk×2.5/GAPDH.

FIG. 37 is a graph showing the expression by quantitative PCR of GATA-4in cells cultured in the presence of DMSO. The vertical axis representsGATA-4/GAPDH.

FIG. 38 is a graph showing the expression by quantitative PCR of α-CA incells cultured in the presence of DMSO. The vertical axis representsα-CA/GAPDH.

FIG. 39 is a graph showing the expression by quantitative PCR of MLC incells cultured in the presence of DMSO. The vertical axis representsMLC/GAPDH.

FIG. 40 is a graph showing the expression by quantitative PCR of MHC incells cultured in the presence of DMSO. The vertical axis representsMHC/GAPDH.

FIG. 41 is a strong magnification image of a sheet containingADMPC-derived cardiac myoblasts.

FIG. 42 is an echocardiograph two weeks before grafting, beforegrafting, and four and 16 weeks after grafting of a sheet containingADMPC-derived cardiac myoblast. The top row is from MI controls and thebottom row is from a heart grafted with a sheet containing ADMPC-derivedcardiac myoblast.

FIG. 43 is an echocardiograph two weeks before grafting, beforegrafting, and four and 16 weeks after grafting of a sheet containingADMPC. The top row is from MI controls and the bottom row is from aheart grafted with a sheet containing ADMPC.

FIG. 44 is a graph (unit of the vertical axis: mm) showing improvementof LVDd of a heart grafted with a sheet containing ADMPC-derived cardiacmyoblasts (circle) and a sheet containing ADMPC (square).

FIG. 45 is a graph (unit of the vertical axis: mm) showing improvementof LVDs of a heart grafted with a sheet containing ADMPC-derived cardiacmyoblasts (circle) and a sheet containing ADMPC (square).

FIG. 46 is a graph showing improvement of % EF of a heart grafted with asheet containing ADMPC-derived cardiac myoblasts (circle) and a sheetcontaining ADMPC (square).

FIG. 47 is a graph showing improvement of % FS of a heart grafted with asheet containing ADMPC-derived cardiac myoblasts (circle) and a sheetcontaining ADMPC (square).

FIG. 48 is a graph showing improvement of LVDs of a heart grafted with asheet containing ADMPC-derived cardiac myoblasts (triangle) incomparison to a sheet containing ADMPC (square).

FIG. 49 is a graph showing improvement of % EF of a heart grafted with asheet containing ADMPC-derived cardiac myoblasts (triangle) incomparison to a sheet containing ADMPC (square).

FIG. 50 is a HE staining image (100×) of a heart grafted with a sheetcontaining cardiac myoblast.

FIG. 51 is a HE staining image (100×) of a heart grafted with a sheetcontaining ADMPC.

FIG. 52 is a micrograph (100×) of a heart grafted with a sheetcontaining cardiac myoblast when immunostained using an anti-human α-CAantibody.

FIG. 53 is a micrograph (100×) of a heart grafted with a sheetcontaining ADMPC when immunostained using an anti-human α-CA antibody.

FIG. 54 is a micrograph (100×) of a heart grafted with a sheetcontaining cardiac myoblast when immunostained using an anti-human MHCantibody.

FIG. 55 is a micrograph (100×) of a heart grafted with a sheetcontaining ADMPC when immunostained using an anti-human MHC antibody.

FIG. 56 is a figure showing micrographs of structures of hearts aftergrafting of sheets containing ADMPC-derived cardiac myoblast and ADMPCwhen immunostained using an anti-human α-CA antibody.

FIG. 57 is a figure showing micrographs of structures of hearts aftergrafting of sheets containing ADMPC-derived cardiac myoblast and ADMPCwhen immunostained using an anti-human MHC antibody.

FIG. 58 is a figure representing the thickness of anti-human α-CAantibody-positive regions after grafting sheets containing ADMPC-derivedcardiac myoblast and ADMPC.

FIG. 59 is a figure graphed with the thickness of anti-human α-CAantibody-positive regions after grafting sheets containing ADMPC-derivedcardiac myoblast and ADMPC as the index.

FIG. 60 is a micrograph (100×) of a heart grafted with a sheetcontaining cardiac myoblast when immunostained with an anti-humanHLA-ABC antibody.

FIG. 61 is a micrograph (100×) of a heart grafted with a sheetcontaining ADMPC when immunostained with an anti-human HLA-ABC antibody.

FIG. 62 is a figure comparing the states of differentiation intoadipocytes for ADMPC and ADSC by oil red 0 staining.

FIG. 63 is a graph showing the content in lipids contained in anadipocyte obtained by causing ADMPC to differentiate. The vertical axisrepresents lipid content (oil red 0 content/well).

FIG. 64 is a figure comparing the states of differentiation into bonesfor ADMPC and ADSC by alizarin red staining.

FIG. 65 is a figure comparing the states of differentiation into bonesfor ADMPC and ADSC by alkaline phosphatase activity.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to a method for obtaining ahepatic-lobule-like cell cluster from an adipose tissue-derived cell,comprising culturing adipose tissue-derived cells. The present inventionis exceptionally excellent on the point that, a cell population similarto a hepatic lobule, which is the minimum functional unit of the liver,can be produced. Adipose tissue-derived cells refers to cells or cellpopulation obtained from a visceral adipose tissue or a subcutaneousadipose tissue, or to cells or cell population induced to differentiatefrom stem cells such as mesenchymal hepatocytes[4] and ES cells andsimilar to cells contained in an adipose tissue in an organism.Normally, adipose tissue-derived cells refer to any or all of adiposetissue-derived stem cells, adipose tissue-derived interstitial cells,adipose tissue-derived multipotent progenitor cells which is describedlate, adipose progenitor cells or cells similar to these, or a cellpopulation containing a mixture comprising all or a portion thereof.Adipose tissue-derived cell can be obtained from adipose tissues and thelike by means/method well known to those skilled in the art. Inaddition, the obtained adipose tissue-derived cell may be grown usingmeans/method well known to those skilled in the art, for instance, tostabilize the phenotype. The adipose tissue-derived cells may be grownby culturing the adipose tissue-derived cells in a culture mediumcontaining dexamethasone and ascorbic acid, for instance, in a 60% DMEM(low glucose) and 40% MCDB201 culture medium added with ITS (10.0 mg/Linsulin, 5.5 mg/L transferrin, 6.7 ng sodium selenite), 1 nMdexamethasone, 0.1 mM ascorbic acid, 10 ng/mL rhEGF, and 5% FCS, in anincubator such as fibronectin-coated dish.

The animal species from which the adipose tissue-derived cell is derivedare not limited in particular, and are preferably, for instance, mammalsincluding mouse, rat, rabbit, dog, cat, cow, horse, monkey and the like,and more preferably human. It is more desirable to use an adiposetissue-derived cell from an identical animal species or an identicalindividual to the animal species in which a disease is to be preventedor treated using the obtained hepatic-lobule-like cell cluster. Sinceadipose tissues are present in sufficient quantities in organisms andare obtained relatively readily, the present invention is exceptionallyexcellent compared to, for instance, methods in which hepatic lobulesare obtained from limited materials such as corpses, and the like. Forinstance, by carrying out the above-mentioned method of the presentinvention using self-derived adipose tissue-derived cells andautografting the obtained hepatic-lobule-like cell cluster, diseasesthat occur due to a decrease in liver functions, such as hepaticcirrhosis, can be treated without fearing a rejection reaction.

Hepatic-lobule-like cell cluster refers to hepatic lobule inside anorganism and to cell population having similar function/morphologythereto, including, for instance, hepatocyte, biliary tract epithelialcell, endothelial cell, Kupffer cell, hepatic stellate cell and thelike. Compared to individual hepatocytes, the hepatic-lobule-like cellcluster of the present invention is exceptionally excellent on the pointthat a sufficient quantity of secretory protein can be produced, thepoint that it has high metabolic capability, on the point that it hashigh detoxification capability, and the like. In addition, it also hasthe advantage of being easily used, for example, in purposes such asgrafting or the like, by causing a cell population to be formed.

It is desirable that the acquisition method of hepatic-lobule-like cellcluster from adipose tissue-derived cell of the present inventioncomprises as an important step the step of causing a hepatic-lobule-likecell cluster to be formed from an undifferentiated cell. It is desirablethat the acquisition method of hepatic-lobule-like cell cluster fromadipose tissue-derived cell of the present invention further comprisesas an important step the step of obtaining an undifferentiated cell fromadipose tissue-derived cells. These steps may be carried out one afterthe other or may be carried out in parallel.

Undifferentiated cell refers to a cell that is capable ofdifferentiating into diverse cells, for instance, hepatic progenitorcell, pancreatic progenitor cell, cardiac muscle progenitor cell,vascular endothelial progenitor cell, osteoblast, chondroblast and thelike. The step of obtaining an undifferentiated cell may furthercomprise the step of growing the obtained undifferentiated cell. Bygrowing the undifferentiated cell, increasing the formation efficiencyof the hepatic-lobule-like cell cluster, increasing the number ofhepatic-lobule-like cell cluster, and the like, are possible. The stepof obtaining the undifferentiated cell may be carried out using knownmethods such as, for instance, sorting, MACS, method by antigen-antibodyreaction such as rosette forming method, density gradient method, methodof selecting based on morphology and single cell cloning, or may becarried out by culturing the adipose tissue-derived cell in a suspendedstate, causing an adiposphere to be formed. Since it allows alreadydifferentiated cell to be killed and undifferentiated cell tosurvive/grow, culturing the adipose tissue-derived cell in a suspendedstate, causing an adiposphere to be formed, is preferred. Regardingculturing in a suspended state, a description will follow. Here, anadiposphere is defined as a spheroid containing undifferentiated cell asthe principal constituent. Since formation of adiposphere and thesubsequent differentiation into a hepatic-lobule-like cell cluster mayoccur sequentially or coincidentally, an adiposphere may contain inaddition to undifferentiated cell, hepatocyte, biliary tract epithelialcell, endothelial cell, Kupffer cell, hepatic stellate cell and thelike.

The step of obtaining hepatic-lobule-like cell cluster is carried out byculturing undifferentiated cell in a culture medium containing, forinstance, fibroblast growth factor, hepatocyte growth factor, oncostatinM, epithelial growth factor, and dimethylsulfoxide; preferably, such asstep is carried out by culturing the undifferentiated cell in asuspended state. Culturing in suspension allows a cell population havinga similar morphology to a hepatic lobule inside an organism to beobtained more readily. Culture of undifferentiated cell in suspendedstate means placing and culturing the cell in a freed state bypreventing or suppressing adhesion to the culture container. Suspensionof a cell can be carried out by a variety of well known means/methods.For instance, cells may be placed in a suspended state using a culturecontainer or apparatus treated to prevent or suppress adhesion of cellsor made with such materials that prevent or suppress adhesion of cells.As culture container or apparatus, low binding culture containers or thelike exist, such as siliconized culture container (for instance,siliconized flask) or low binding culture dish (for instance, HydroCell(CellSeed)). Or cells may be cultured in a suspended state using thehanging drop culture method. In addition, well known means/method may beused in combination suitably at suspension starting time point or tocontinue suspension. As examples of such means/method, freeing cellswith an enzyme or a chelator such as trypsin/EDTA, collagenase and CellDissociation Buffer (GIBCO Invitrogen), scraping off cells physicallyusing a scraper] or the like, or methods whereby cells are cultured witha temperature responsive culture equipment[5] for cell recovery (forinstance, RepCell (CellSeed)), then, detaching the cells by incubationat for instance, 20° C. for 30 minutes, and the like, exist. Theabove-mentioned hepatic-lobule-like cell cluster is formed by culturingundifferentiated cell in a suspended state.

In another aspect, the present invention relates to ahepatic-lobule-like cell cluster obtainable by the above-mentionedmethod. As described above, the hepatic-lobule-like cell cluster of thepresent invention contains a hepatocyte. For instance, carrying out theabove method using cells collected from the adipose tissue of a subject,or a similar to the subject, having a disease that occurs due to adecrease in liver function such as hepatic cirrhosis or predispositiontherefor, and grafting the obtained hepatic-lobule-like cell cluster tothe subject allow diseases that occur due to a decrease in liverfunction such as hepatic cirrhosis to be treated or prevented, and thelike.

In a further aspect, the present invention relates to hepatocytecontained in the hepatic-lobule-like cell cluster obtainable by theabove method.

In an even further aspect, the present invention relates to a medicinalcomposition for preventing or treating a disease that occurs due to adecrease in liver function, containing a hepatic-lobule-like cellcluster that obtainable by the above method and/or a hepatocytecontained in such a hepatic-lobule-like cell cluster. A disease thatoccurs due to a decrease in liver function includes, disease that occursdue not only to a decrease but also to insufficiency in liver function,for instance, hepatitis, hepatic cirrhosis, liver cancer, hepaticinsufficiency, drug liver damage, alcoholic liver damage, congenitalmetabolic anomaly, cholestatic liver damage and the like. In themedicinal composition of the present invention, the hepatic-lobule-likecell cluster or the hepatocyte may be suspended in a suitable solutionsuch as PBS. Also, the medicinal composition of the present inventionmay contain, in addition to the hepatic-lobule-like cell cluster orhepatocyte, a substance that promotes grafting thereof to liver, liverfunction improvement drug, suitable additive, diluent and the like.

In another further aspect, the present invention relates to use ofhepatic-lobule-like cell cluster obtainable by the above method and/orhepatocyte contained in such a hepatic lobule cell population, for thepreparation of a medicinal product for preventing or treating a diseasethat occurs due to a decrease in liver function.

In a different aspect, the present invention relates to a method for thetreatment or prevention of a disease that occurs due to a decrease inliver function comprising administering a subject with thehepatic-lobule-like cell cluster obtainable by the above-mentionedculture method and/or hepatocyte contained in such a hepatic-lobule-likecell cluster. From the point of rejection reaction and the like, ahepatic-lobule-like cell cluster or hepatocyte obtainable from anidentical species or autologous adipose tissue-derived cell is usedpreferably in the present invention. Hepatic-lobule-like cell cluster orhepatocyte may be grafted or injected, for instance, under the renalcapsule[6], via portal vein, inside the liver, inside the great omentum,side the peritoneal cavity, inside the spleen, under the skin and thelike. The subject may be any one; it may be a human subject, or it maybe a subject other than human, for instance, a mammal such as mouse ormonkey. The administration quantity, administration frequency and thelike of the hepatic-lobule-like cell cluster or hepatocyte are selectedsuitably according to a variety of factors such as, for instance, thestate of the subject, and the degree of seriousness of the disease.

In addition, the present invention relates to a method for decreasingthe blood concentration of bilirubin comprising administering a hepaticlobule cell population and/or a hepatocyte contained in such ahepatic-lobule-like cell cluster. Such a method may be performed eitherin vitro or in vivo.

In another further aspect, the present invention relates to a method ofscreening for a substance that promotes formation of hepatic lobule,comprising adding a candidate substance to the culture medium whenculturing an adipose tissue to obtain a hepatic-lobule-like cellcluster, and showing that the candidate substance is a substance thatpromotes formation of hepatic lobule when formation ofhepatic-lobule-like cell cluster has been promoted compared to formationin a system not containing the candidate substance. As described above,since a hepatocyte is contained in the hepatic-lobule-like cell cluster,such a method also comprises a method of screening for a substance thatpromotes differentiation into hepatocyte. As candidate substances, manyexist and, for instance, analogs or derivatives of basic fibroblastgrowth factor, hepatocyte growth factor or oncostatin M, and the like,may be cited, without being limited to these. The addition of acandidate substance to the culture medium when obtaining ahepatic-lobule-like cell cluster from an adipose tissue may be carriedout once or several times at either or both of the step of obtaining anundifferentiated cell from an adipose tissue-derived cell and the stepof obtaining a hepatic-lobule-like cell cluster from an undifferentiatedcell.

The formation of hepatic-lobule-like cell cluster can be checked, forinstance, by measuring the number of formed hepatic-lobule-like cellclusteres by microscopic observation, by quantifying α-fetoprotein,albumin and the like secreted in the culture supernatant, for instance,using ELISA, by measuring the expression of genes such as ofα-fetoprotein, albumin, CYP1B1, glutamine synthase, keratin 18 andkeratin 19 by quantitative PCR, or by measuring a marker substance ofwhich the expression is known to decrease [7] or increase accompanyingthe differentiation into/formation of hepatic lobule, for instance,transthyretin, α1-anti-trypsin, tyrosine aminotransferase,glucose-6-phosphatase and the like, by quantitative PCR or ELISA or thelike.

Consequently, in a further aspect, the present invention relates to asubstance obtainable by the above-mentioned screening method, whichpromptes formation of hepatic lobule. The number of the obtainedhepatic-lobule-like cell clusteres may be increased, or the speed offormation of hepatic-lobule-like cell cluster may be increased, by usingsuch a substance in the method for obtaining a hepatic-lobule-like cellcluster from an adipose tissue-derived cell of the present invention.Or, such a substance may be used in the treatment or prevention of adisease that occurs due to a decrease in liver function.

Another further aspect of the present invention relates to a method ofscreening for a substance that inhibits formation of hepatic lobule,comprising adding a candidate substance to the culture medium whenculturing an adipose tissue-derived cell to obtain a hepatic-lobule-likecell cluster, and showing that the candidate substance is a substancethat inhibits formation of hepatic lobule when formation ofhepatic-lobule-like cell cluster has been inhibited compared toformation in a system not containing the candidate substance. Asdescribed above, since a hepatocyte is contained in thehepatic-lobule-like cell cluster, such a method also comprises a methodof screening for a substance that inhibits differentiation intohepatocyte. As candidate substances, many exist and, for instance,analogs or derivatives of drugs having hepatotoxicity such as carbontetrachloride and phenobarbital, and the like, may be cited, withoutbeing limited to these. A substance obtainable by such a screeningmethod is sought to be suited for the treatment or prevention of adisease occurring due to liver hyperfunction. Regarding addition of thecandidate substance to the culture medium and means/method forevaluating formation of hepatic-lobule-like cell cluster are asdescribed above.

Consequently, in a further aspect, the present invention relates to asubstance obtainable by the above-mentioned screening method, whichinhibits formation of a hepatic lobule.

In yet another further aspect, the present invention relates to a kitused in the above-mentioned method of screening for a substance thataccelerates or suppresses the formation of a hepatic lobule. The kit ofthe present invention may contain cell acquisition means from an adiposetissue, a culture medium, a culture container, as well as means forchecking the formation of the hepatic-lobule-like cell cluster, and thelike. Normally, handling instructions are included with the kit. Usingsuch a kit allows the above-mentioned screening to be carried outrapidly and readily.

In another aspect, the present invention relates to a method ofscreening for a substance that elevates the activity of a hepaticlobule, comprising culturing in a medium containing a candidatesubstance a hepatic-lobule-like cell cluster obtained by culturing anadipose tissue-derived cell, and showing that the candidate substance isa substance that elevates the activity of a hepatic lobule when theactivity of the hepatic-lobule-like cell cluster has been increasedcompared to the activity in a system not containing the candidatesubstance. Activity of a hepatic lobule refers to detoxification action,protein synthesis capability, metabolic action, and the like, of thehepatic lobule.

Elevation of the activity of a hepatic-lobule-like cell cluster can bechecked, for instance, by quantifying α-fetoprotein, albumin and thelike secreted in the culture supernatant using ELISA or the like, andfrom the increase or decrease of such protein quantities, or bymeasuring the expression of genes such as of α-fetoprotein, albumin,CYP1B1, glutamine synthase, keratin 18 and keratin 19 by quantitativePCR, and from the increase or decrease of the expression of such genes.As candidate substances, many exist and, for instance, analogs orderivatives of basic fibroblastic growth factor, hepatocyte growthfactor, or oncostatin M, and the like, may be cited, without beinglimited to these. Addition of candidate substance into the culturemedium may be carried out once of multiple times.

Consequently, in another aspect, the present invention relates to asubstance obtainable by the above-mentioned screening method, whichelevates the activity of a hepatic lobule. The activity of the obtainedhepatic-lobule-like cell cluster may be increased by using such asubstance in the method for obtaining a hepatic-lobule-like cell clusterfrom an adipose tissue-derived cell of the present invention. Or, such asubstance may be used in the treatment or prevention of a disease thatoccurs due to a decrease in liver function.

In another aspect, the present invention relates to a method ofscreening for a substance that diminishes the activity of a hepaticlobule, comprising culturing in a medium containing a candidatesubstance a hepatic-lobule-like cell cluster obtained by culturing anadipose tissue-derived cell, and showing that the candidate substance isa substance that diminishes the activity of a hepatic lobule when theactivity of the hepatic-lobule-like cell cluster has been diminishedcompared to the activity in a system not containing the candidatesubstance. As candidate substances, many exist and, for instance, carbontetrachloride an phenobarbital, and the like, may be cited, withoutbeing limited to these. Regarding addition of the candidate substanceinto the culture medium, means/method for evaluating the activity ofhepatic-lobule-like cell cluster, the descriptions are as above.

Consequently, in another aspect, the present invention relates to asubstance obtainable by the above-mentioned screening method, whichdiminishes the activity of a hepatic lobule.

In yet another further aspect, the present invention relates to a kitused in the above-mentioned method of screening for a substance thatelevates or diminishes the activity of a hepatic lobule. The kit of thepresent invention may contain cell acquisition means from an adiposetissue, a culture medium, a culture container, as well as means forchecking the activity of the hepatic-lobule-like cell cluster, and thelike. Normally, handling instructions are included with the kit. Usingsuch a kit allows the above-mentioned screening to be carried outrapidly and readily.

In one aspect, the present invention relates to a cell populationcontaining an adipose tissue-derived multipotent progenitor cell.Adipose tissue-derived multipotent progenitor cell refers to a cell,which is a cell that can differentiate into a variety of cell lines suchas of the endoderm, mesoderm and ectoderm, and expressing Islet-1, amarker for the absence of differentiation. An adipose tissue-derivedmultipotent progenitor cell can be obtained from an embryonic stem cellor the like by causing differentiation, in addition to an adiposetissue. The animal species from which the adipose tissue-derivedmultipotent progenitor cell is derived are not limited in particular,and are preferably, for instance, mammals including human, mouse, rat,rabbit, dog, cat, cow, horse, monkey and the like, and more preferablyhuman. Or, the species similar to or identical to a subject to betreated by a regenerative medical therapy using such a cell populationis desirable.

Since the cell population of the present invention has a low proportionof undesired contaminants, for instance, cells other than the adiposetissue-derived multipotent progenitor cell, such as erythrocytes andvascular endothelial cells, it has advantages such as ease of cultureand high differentiation efficiency. As measures to eliminate suchcontaminants, means/methods using the difference with the specificgravity adipose tissue-derived multipotent progenitor cell, forinstance, the density method, means/methods using the difference withthe adhesiveness of the adipose tissue-derived multipotent progenitorcell, methods using for instance a chelator such as EDTA or an enzymesuch as trypsin, antigen-antibody method such as sorting and MACS,methods that select based on morphology, single cell cloning, hemolysismethod, and the like, may be cited. The decrease in contaminants withinthe cell population may be verified, for instance, by quantifying amarker that the contaminant has using methods such as RT-PCR and ELISA,visually under a microscope, or by flow cytometry or immunohistologicalstaining.

The cell population of the present invention preferably contains atleast 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96% or 99%adipose tissue-derived multipotent progenitor cell. Inclusion of adiposetissue-derived multipotent progenitor cells in such proportions givesthe cell population of the present invention advantages such as ease ofmaintenance of the adipose tissue-derived multipotent progenitor cellsand high efficiency when [they are] caused to differentiate. In additionto the adipose tissue-derived multipotent progenitor cells, the cellpopulation of the present invention may contain cells that are effectivefor the maintenance or differentiation of the adipose tissue-derivedmultipotent progenitor cells, such as, feeder cells, vascularendothelial cells, fibroblasts and the like. Inclusion of such cells mayenhance the above advantages.

In another aspect, the present invention relates to a method, which is amethod for obtaining an adipose tissue-derived multipotent progenitorcell from an adipose tissue, comprising the steps of (a) removingerythrocytes from an adipose tissue-derived cell population to obtain apreadipose tissue-derived multipotent progenitor cell population, andnext, (b) removing cells other than an adipose tissue-derivedmultipotent progenitor cell from the preadipose tissue-derivedmultipotent progenitor cell population to obtain an adiposetissue-derived multipotent progenitor cell. The present invention allowsadipose tissue-derived cells other than the adipose tissue-derivedmultipotent progenitor cells to be decreased, and the adiposetissue-derived multipotent progenitor cells to be obtained at high yieldand high purity. In the present invention, the above steps may becarried out one after the other, or may be carried out in parallel. Theadipose tissue used in this aspect of the present invention may beeither of a subcutaneous adipose tissue and a visceral adipose tissuefrom an organism. The animal species from which the adipose tissue isderived are not limited in particular, and are preferably, for instance,mammals including human, mouse, rat, rabbit, dog, cat, cow, horse,monkey and the like, and more preferably human. Or, the species similarto or identical to the subject to be treated by a regenerative medicaltherapy using the adipose tissue-derived multipotent progenitor cellobtainable by the method of the present invention is desirable.

The adipose tissue-derived cell population used herein refers to a cellpopulation containing at least an adipose tissue-derived multipotentprogenitor cell. The adipose tissue-derived cell population may contain,in addition to an adipose tissue-derived multipotent progenitor cell,erythrocytes, vascular endothelial cells, fibroblasts and the like. Theadipose tissue-derived cell population is obtained by treating anadipose tissue, for instance, with an enzyme such as collagenase, or byphysical means/method, and/or eliminating lipids and the like, forinstance, by centrifugal separation and filtration.

Erythrocytes have the properties of adsorbing adipose tissue-derivedmultipotent progenitor cells, whereby the yield of adiposetissue-derived multipotent progenitor cell may be decreased.Consequently, it is necessary to eliminate erythrocytes from the adiposetissue-derived cell population. Elimination of erythrocytes from theadipose tissue-derived cell population may be carried out by anymeans/method, for instance, it may be one carried out by a means/methodother than one based on the difference in adhesiveness between theerythrocytes and cells other than these. Preferably, such elimination iscarried out by the density method, the hemolysis method or thefiltration method, and more preferably, by the density method. Thedensity method may be carried out using a density solution with anadequate density, for instance, a commercially available densitysolution such as Lymphoprep. It suffices that the density of the densitysolution used is one between the densities of erythrocytes and cellsother than these, preferably 1.063 to 1.119, more preferably 1.070 to1.110 and most preferably 1.077.

The preadipose tissue-derived multipotent progenitor cell populationused herein refers to a cell population containing at least an adiposetissue-derived multipotent progenitor cell. The preadiposetissue-derived multipotent progenitor cell population may contain, inaddition to the adipose tissue-derived multipotent progenitor cell,vascular endothelial cells, fibroblasts and the like. As mentionedabove, the preadipose tissue-derived multipotent progenitor cellpopulation, in substance, does not contain erythrocytes. By eliminatingthe erythrocytes to produce a preadipose tissue-derived multipotentprogenitor cell population, the subsequent elimination of cells otherthan the adipose tissue-derived multipotent progenitor cell may becarried out readily and efficiently.

The cells other than the adipose tissue-derived multipotent progenitorcell used herein refer to adherent cells, or the like, such as vascularendothelial cells and fibroblasts. Although elimination of cells otherthan the adipose tissue-derived multipotent progenitor cells from thepreadipose tissue-derived multipotent progenitor cell population may becarried out by any means/methods, it is carried out using preferably asubstance other than trypsin, more preferably a chelator such as EDTA orEGTA and most preferably EDTA. Preferably, such elimination is one thatis carried out based on the difference in adhesiveness between theadipose tissue-derived multipotent progenitor cells and cells other thanthese. In addition to the above, cells other than adipose tissue-derivedmultipotent progenitor cells can be eliminated, for instance, by filterfiltration[3] or the like. the purity and yield of the obtained adiposetissue-derived multipotent progenitor cell population increases byeliminating these cells.

In another aspect, the present invention relate to an adiposetissue-derived multipotent progenitor cell obtainable by the method forobtaining an adipose tissue-derived multipotent progenitor cell from anadipose tissue described above. Such an adipose tissue-derivedmultipotent progenitor cell expresses Islet-1, as described above.

In a further aspect, the present invention relates to a cell populationcontaining an adipose tissue-derived multipotent progenitor cellobtainable by the method for obtaining an adipose tissue-derivedmultipotent progenitor cell from an adipose tissue described above. Sucha cell population, in substance, does not contain undesirable adiposetissue-derived cells such as erythrocytes and vascular endothelialcells, but may contain cells that are effective for themaintenance/differentiation and the like of the adipose tissue-derivedmultipotent progenitor cells, such as, feeder cells.

In addition, cardiac myoblasts are provided by the present invention insufficient amounts to constitute a sheet. Consequently, in one aspect,the present invention provides a sheet that contains cardiac myoblasts.Herein, a cardiac myoblast refers to a cell that has been directed todifferentiate into a cardiac myocyte, which is a cell expressingα-cardiac actin (α-CA) and Myosin Light Chain (MLC). The animal speciesfrom which the cardiac myoblast is derived are not limited inparticular, and are preferably, for instance, mammals including human,mouse, rat, rabbit, dog, cat, cow, horse, monkey and the like, and morepreferably human. Or, the species similar to or identical to the subjectin which such sheet containing cardiac myoblasts is applied isdesirable. For instance, by grafting a subject with a sheet containingcardiac myoblasts derived from the same species or the same animal asthe subject, treating severe cardiac failure, or the like, becomespossible, without fearing a rejection reaction.

A sheet containing cardiac myoblasts refers to a cell populationcontaining cardiac myoblasts as essential constituent. In the sheet, thecardiac myoblast may be contained in either form of a mono-layer or amulti-layer. Having the morphology of a sheet allows for ease ofhandling when used for grafting or the like. Constituents of the sheetother than cardiac myoblasts may be any, for instance, adiposetissue-derived stem cells, cardiac myocytes, cell scaffolds, vascularendothelium, matrix, and the like, may be cited. The size and thicknessof the sheet may be selected suitably according to a variety ofconditions such as the extent of the injured area.

The proportion of cardiac myoblasts contained in a sheet is not limitedin particular and may be selected suitably according to a variety ofconditions such as, for instance, the state of the subject in which thesheet is applied. The proportion is, for instance, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% and the like. The proportion ofcardiac myoblasts may be determined by quantifying α-CA or MLC, whichare cardiac myoblast marker genes, using means known to those skilled inthe art, for instance, quantitative RT-PCR.

The function of the sheet of the present invention can be checked bywell known methods, for instance, by grafting a sheet to a subject, andby echography of the cardiac function of the grafted subject, or bymeasuring the diameter at end-diastole (LVDd), the diameter atend-systole (LVDs), the left ventricular ejection fraction (% EF) or theleft ventricular internal diameter shortening fraction (% FS) and thelike.

In another aspect, the present invention provides a method for obtainingcardiac myoblasts, comprising culturing an adipose tissue-derived stemcell. According to the method, cardiac myoblasts can be obtained inlarge amounts from an adipose tissue-derived stem cell. The method maycomprise the step of obtaining an adipose tissue-derived stem cell froman adipose tissue-derived cells. An adipose tissue-derived stem cellrefers to a cell, which is a cell that can differentiate into a varietyof cell lines such as of the endoderm, mesoderm and ectoderm, andincludes adipose tissue-derived multipotent progenitor cell (ADMPC),which expresses Islet-1, a marker for the absence of differentiation.The animal species from which the adipose tissue-derived stem cell isderived are not limited in particular, and are preferably, for instance,mammals including human, mouse, rat, rabbit, dog, cat, cow, horse,monkey and the like, and more preferably human. Or, the species similarto or identical to a subject to be treated with cardiac myoblastsobtained from such adipose tissue-derived stem cell is desirable.

Preferably, the method for obtaining cardiac myoblasts of the presentinvention is one that comprises the step of culturing an adiposetissue-derived stem cell in the presence of a DMSO or OP9 culturesupernatant. By culturing an adipose tissue-derived stem cell under suchcondition, the cell may differentiate and/or be induced into a cardiacmyoblast. The culture medium used in such culture may be selectedsuitably. Such a culture medium may be one containing a variety offactors such as, for instance, retinoic acid, BMP2, BMP4, TGFβ2, HGF,bFGF, thyroxine, oxytocin or fatty acid concentrate. Differentiationinto cardiac myoblast can be checked by measuring the expression of acardiac myoblast marker such as, for instance, α-CA or MLC by RT-PCR.

Consequently, in a further aspect, the present invention providescardiac myoblast obtainable by the method for obtaining cardiacmyoblasts described above. Such cardiac myoblasts can be used intreatment of cardiac diseases such as myocardial infarction and cardiacmyopathy, or can be used as materials for a sheet containing cardiacmyoblasts.

In another aspect, the present invention provides a method, which is amethod for obtaining a sheet containing cardiac myoblasts, comprisingthe following steps: (a) causing an adipose tissue-derived stem cell todifferentiate into a cardiac myoblast, then, (b) causing a sheetcontaining cardiac myoblasts to form. These steps may be carried out oneafter the other or may be carried out in parallel. According to such amethod, a sheet containing cardiac myoblasts can be obtained readily andefficiently. The step of causing an adipose tissue-derived stem cell todifferentiate into a cardiac myoblast is as described above.

The step of causing a sheet containing cardiac myoblasts to form from acardiac myoblast may be achieved by means or method known by thoseskilled in the art. Preferably, the step may be achieved by causing acardiac myoblast to multiply in an attached state to form a cellpopulation and then peeling the formed cell population. The number ofcardiac myoblasts used and culture time may be selected suitablyaccording to a variety of conditions, such as, the extent of theobtained sheet and the number of cardiac myoblasts contained in thesheet. For instance, a sheet may be obtained by culturing 10⁵ to 10⁶cardiac myoblasts for 24 to 72 hours. Peeling of the cell population maybe carried out by a variety of means, for instance, physicalstimulation. Or, the cardiac myoblasts may be cultured in atemperature-sensitive culture dish and incubated for instance at 20° C.or below to peel the cell population.

Consequently, in a further aspect, the present invention relates to asheet containing cardiac myoblasts, obtainable by the method forobtaining a sheet containing cardiac myoblasts described above. Such asheet containing cardiac myoblasts can be used in the treatment ofcardiac diseases such as myocardial infarction and cardiac myopathy.

In another aspect, the present invention relates to a method fortreating and/or preventing a disease occurring due to a decline in thefunction of cardiac muscle, comprising grafting a sheet containingcardiac myoblasts or cardiac myoblasts to a subject. Diseases occurringdue to a decline in the function of cardiac muscle are, for instance,acute myocardial infarction, old myocardial infarction, ischemicmyocardial infarction, dilated cardiac myopathy, congenital cardiacdisease and the like. Grafting to a subject may be carried out bymethods known to those skilled in the art. It may be carried out, forinstance, by grafting a sheet containing cardiac myoblasts to a regionwhere function of cardiac muscle decreased, or by grafting cardiacmyoblasts via the coronary artery. The size and thickness of the sheetto be grafted, the number of cardiac myoblasts contained in the sheet,and the number of cardiac myoblasts may be selected suitably accordingto a variety of conditions such as the state of the subject and theextent of the injured region.

Consequently, in another aspect, the present invention provides acomposition containing a sheet containing cardiac myoblasts or cardiacmyoblasts for treating and/or preventing disease caused by a decrease incardiac function. Such a composition may contain, in addition to thesheet or the cardiac myoblasts, for instance, PBS, a culture medium, asubstance that promotes grafting, a cardiac function improver, a growthfactor, a proliferation factor and the like.

In another aspect, the present invention relates to a method, which is amethod of screening for a substance that promotes differentiation intocardiac myoblast, comprising the following steps:

(a) causing an adipose tissue-derived stem cell to differentiate into acardiac myoblast in a culture medium containing a candidate substance;

(b) checking the differentiation of the adipose tissue-derived stem cellinto cardiac myoblast, and showing that the candidate substance is asubstance that promotes differentiation into cardiac myoblast when thedifferentiation has been promoted compared to the differentiation whenthe adipose tissue-derived stem cell is cultured in a culture medium notcontaining the candidate substance. As candidate substances, many existand analogs and derivatives of, for instance, retinoic acid, BMP2, BMP4,TGFβ2, HGF, bFGF, thyroxine, or oxyton[8], and the like, may be cited,without limiting to these. Differentiation of adipose tissue-derivedstem cell into cardiac myoblast is as described above. Differentiationinto cardiac myoblast can be checked by a variety of methods, forinstance, by measuring the expression of a cardiac myoblast marker suchas α-CA or MLC by RT-PCR.

Consequently, in a further aspect, the present invention relates to asubstance that promotes differentiation into cardiac myoblast, which canbe obtained by the method of screening for a substance that promotesdifferentiation into cardiac myoblast described above. The substance maybe used in the production method for the cardiac myoblast and sheetcontaining cardiac myoblasts described above to increase the number ofthe obtained cardiac myoblasts and sheets. Or, such a substance may beused in the treatment or prevention of a disease caused by a decrease incardiac function.

In another aspect, the present invention relates to a method, which is amethod of screening for a substance that inhibits differentiation intocardiac myoblast, comprising the following steps:

(a) causing an adipose tissue-derived stem cell to differentiate into acardiac myoblast in a culture medium containing a candidate substance;

(b) checking the differentiation of the adipose tissue-derived stem cellinto cardiac myoblast, and showing that the candidate substance is asubstance that inhibits differentiation into cardiac myoblast when thedifferentiation has been inhibited compared to the differentiation whenthe adipose tissue-derived stem cell is cultured in a culture medium notcontaining the candidate substance. As candidate substances, many existand analogs and derivatives of, for instance, noggin, and the like, maybe cited, without limiting to these.

Consequently, in a further aspect, the present invention relates to asubstance that inhibits differentiation into cardiac myoblast, which canbe obtained by the method of screening for a substance that inhibitsdifferentiation into cardiac myoblast described above. The substance maybe used in the treatment or prevention of a disease caused by anelevation in cardiac function.

In addition, the present invention relates to a kit for screening for asubstance that promotes or inhibits differentiation into cardiacmyoblast. The kit of the present invention may contain an adiposetissue-derived stem cell, a culture medium, a culture container, as wellas means for checking differentiation into cardiac myoblast and thelike. Normally, handling instructions are included with the kit. Usingsuch a kit allows the above-mentioned screening to be carried outrapidly and readily.

In another aspect, the present invention provides a method, which is amethod of screening for a substance that promotes or inhibits formationof a sheet containing cardiac myoblasts, comprising the following steps:

(a) causing an adipose tissue-derived stem cell to differentiate into acardiac myoblast in a culture medium containing a candidate substance;or

(b) causing a sheet containing cardiac myoblasts to formation in thepresence of a candidate substance;

(c) checking the formation of a sheet containing cardiac myoblasts,

and showing that the candidate substance is a substance that promotes orinhibits formation of a sheet containing cardiac myoblasts when theformation has been promoted or inhibited compared to that formed in asystem not containing the candidate substance. The method for checkingthe formation of sheet is as described above. The addition of candidatesubstance may be carried out in both steps (a) and (b) described above,or may be carried out in either one.

Consequently, in a further aspect, the present invention relates to asubstance that promotes or inhibits formation of a sheet containingcardiac myoblasts, which can be obtained by the screening method for asubstance that promotes or inhibits formation of a sheet containingcardiac myoblasts described above.

In addition, the present invention relates to a kit for screening for asubstance for promoting or inhibiting formation of a sheet containingcardiac myoblasts described above.

Hereinafter, the present invention will be described in detail andconcretely showing examples; however the examples do not limit thepresent invention.

Example 1 Isolation and Culture of Adipose Tissue-Derived Cell

A human adipose tissue was sliced into 2 to 3 mm²-large fragments anddigested using collagenase I. The digestate was cultured for 24 to 36hours in DMEM containing 10% FBS and antibiotics and treated with 0.02%EDTA to obtain adipose tissue-derived cells. The obtained adiposetissue-derived cells were amplified by 3 to 5 passage cultures in aculture medium containing 60% DMEM (low glucose), 40% MCDB201, 1×ITS(10.0 mg/L insulin, 5.5 mg/L transferrin, 6.7 ng sodium selenite), 10ng/mL rhEGF, 1 nM dexamethasone, 0.1 mM ascorbic acid and 5% FCS(Hyclone), in a fibronectin-coated dish. A micrograph of adiposetissue-derived cells is shown in FIG. 1.

Acquisition of Undifferentiated Cell from Adipose Tissue-Derived Cells

Cells that were singularized by treating adipose tissue-derived cellstreated with 0.25% trypsin/EDTA (Nacalai Tesque) to be dissociated werecultured for 2 to 3 days in knock out DMEM (GIBCO Invitrogen) containing20% FCS, 1 mM glutamine (GIBCO Invitrogen) and 1% non-essential aminoacids (GIBCO Invitrogen), in a low binding culture dish (Hydrocell;CellSeed). The cells autoagglutinated to form adipospheres. A micrographof adipospheres is shown in FIG. 2.

Formation of Hepatic-Lobule-Like Cell Cluster from Undifferentiated Cell

The obtained adiposphere was washed 2 to 3 times in PBS (centrifugationat 1000 to 1200 rpm) and cultured for 3 to 4 weeks in a culture mediumcontaining 60% DMEM (low glucose), 40% MCDB201, 1×ITS, 1 nMdexamethasone, 10011M ascorbic acid, 10 ng/mL rhEGF, bFGF, HGF and OSM(oncostatin M), in a low binding culture dish (Hydrocell; CellSeed).From day 10 of the beginning of the culture, 0.1% DMSO was added toobtain a hepatic-lobule-like cell cluster. A micrograph of the obtainedhepatic-lobule-like cell cluster is shown in FIG. 3.

Example 2 Hepatocyte Gene Expression Characteristics ofHepatic-Lobule-Like Cell Cluster 1. Extraction of RNA

Extraction of RNA from the hepatic-lobule-like cell cluster was carriedout using RNeasy Protect Mini Kit (QIAGEN), as follows. Thehepatic-lobule-like cell cluster was recovered, and the buffer RLTcontaining 10 μl/ml 2-mercaptoethanol (Naacalai Tesqu[13]) was added ata proportion of 600 μl/10⁷ cell. Cells were homogenized by pipettingwith a 20G needle and then 600 μl of 70% ethanol was added. Transferredonto an RNeay[14] Mini column inside a 2 ml collection tube were 700 μlof the obtained mixed solution, which was centrifuged at 1000 rpm for 15seconds. Next, 350 μl of buffer RW1 was added onto the column andcentrifuged at 1000 rpm for 15 seconds. Added to 70 μl of buffer RDDwere 10 μl of DNase I stock solution (QIAGEN), which were tumble-mixed,added to the RNeasy silica gel membrane inside the RNeasy Mini column,and incubated at room temperature for 15 minutes. Added was 350 μl ofbuffer RW1, and centrifugation was performed at 1000 rpm for 15 seconds.The 2 ml collection tube was replaced with a new one. Added onto thecolumn was 500 μl of buffer RPE, and centrifugation was performed at1000 rpm for 15 seconds. Further, 50 μl of buffer RPE was added,centrifugation was performed at 1000 rpm for 2 minutes, and then,centrifugation was performed at 1500 rpm for one minute. The column wastransferred to a 1.5 ml collection tube, 30 to 50 μl of RNase free waterwas added to the RNeasy silica gel membrane, and centrifugation wasperformed at 1000 rpm for one minute to extract RNA.

2. Preparation of Single-Stranded cDNA

To 11.5 μl of the extracted RNA solution, 0.5 μl of 0.5 mM Random Primer(Invitorogen[15]) and 1 μl of 10 mM dNTPmix (Invitorogen[16]) wereadded, reacted at 65° C. for 5 minutes, and then cooled on ice. To theobtained mixture, 4 μl of 5× First-Strand buffer (Invitorogen[17]), 1 μlof 0.1M DTT (Invitorogen[18]), 1 μl of RNaseOUT (Invitorogen[19]), and 1μl of SuperScript III RT (Invitorogen[20]) were added, reacted at 25° C.for 5 minutes, at 50° C. for 60 minutes and at 70° C. for 15 minutes toprepare a single-stranded cDNA. The obtained cDNA was stored at 4° C.until use.

3. Real-Time PCR

To 9 μl of the cDNA prepared, 10 μl of TaqMan Universal PCR Master Mix(Applied Biosystems) and 1 μl of TaqMan Gene Expression Assays (AppliedBiosystems) were added. Real-time PCR was carried out with AppliedByosytems[21] 7900 Fast Real-Time PCR system, with the followingconditions: denaturation at 95° C. for 10 minutes, 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. The TaqMan probes sued forα-fetoprotein, albumin, CYP1B1, glutamine synthase, keratin 18 andkeratin 19 are shown in Table 1 below. In addition, GAPDH (AppliedBiosystems) was used as an internal standard. Hepatocytes obtained bythe methods described in Seo M J. et al., Biochem Biophys Res Commun.2005 Mar. 4; 328 (1):258-64 (indicated by “conventional methods”),fibroblasts, HepG2, and adipose tissue-derived cells (represented by“ADSC”) were used as control samples. The results are shown in FIGS. 4to 9 (the vertical axes in the figures represent fluorescenceintensities). In comparison to the hepatocytes obtained by conventionalmethods, the hepatic-lobule-like cell cluster obtained by methods of thepresent invention was found to express α-fetoprotein, albumin, keratin18, keratin 19, CYP1B1 and glutamine synthase highly. In addition, theexpression of these genes excepting albumin was significantly higherthan that in HepG2.

TABLE 1 Gene name Reference sequence Assay ID α-fetoprotein× NM_001134Hs00173490_m1 Albumin NM_000477 Hs00609411_m1 Cytochrome P450, NM_000104Hs00164383_m1 family 1, Subfamily B, polypeptide 1 (CYP1B1) Glutaminesynthase NM_002065 Hs00374213_m1 Keratin 18 NM_199187/NM_000224Hs01941416_g1 Keratin 19 NM_002276 Hs00761767_s1

Example 3 Production of Hepatic Protein by Hepatic-Lobule-Like CellCluster A. Western Blot Analysis 1. Sample Preparation

Hepatic-lobule-like cell cluster was washed three times with PBS(Nacalai Tesque), and then M-PER (PIERCE) was added. Cells were lysed byultrasonication, centrifuged at 14000 g for 15 minutes to eliminateinsoluble cell constituents. Sample buffer (Nacalai Tesque) was added inthe same amounts as the sample, boiled at 100° C. for 5 minutes andice-cooled. The protein concentration in the obtained sample wasmeasured using BCA Protein Assay Reagent (PIERCE).

2. SDS-PAGE

Gel mini plate for electrophoresis (PAG mini “Daiichi”; Daiichi PureChemicals Co.) and running buffer were used to perform SDS-PAGE. Theamount of protein used was 5 μg. The electrophoresis conditions were 10mA in the stacking gel and 40 mA in the running gel.

3. Western Blot

The electrophoresed gel above was washed in blotting buffer for 10minutes. Next, the proteins in the gel were copied onto a nitrocellulosemembrane by wet blotting (100 mA, overnight).

4. Immunostaining

The membrane was washed for 10 minutes in PBS containing 0.1% Tween20.Blocking One (Nacalai Tesque) was used and reacted at room temperaturefor one hour. Reaction was performed for one hour with a 500-folddiluted solution of Human Albumin antibody (BETHYL) or Alpha FetoproteinAb-2 (LAB Vision) as primary antibody. Washing was performed for 15minutes in PBS containing 0.1% Tween20 (three times). Reaction wasperformed for one hour with a 1000-fold diluted solution of polyclonalpig anti-rabbit immunoglobulin/HRP or polyclonal rabbit anti-goatimmunoglobulin/HRP as secondary antibody. Washing was performed for 15minutes in PBS containing 0.1% Tween20 (three times). The bands weredetected using ECL Plus Western Blotting Detection Reagents. The resultsare shown in FIGS. 10 and 11. The hepatic-lobule-like cell clusterobtained by methods of the present invention was found to producesufficient amounts of α-fetoprotein and albumin.

B. Immunohistochemical Staining 1. Preparation of Sections

The hepatic-lobule-like cell cluster was washed three times was PBS(Nacali[22] Tesque) and centrifuged. The obtained pellet was embedded inTissue-Tek OCT-compound (Sakura Fineteck Inc.) and conserved at −30° C.Using a cryostat, 7 μm sections were prepared, pasted on glass andconserved at −30° C.

2. Immunohistochemical Staining

The sections described above were dried with a drier and fixed in 4%paraformaldehyde for 30 minutes. Washing in PBS for 5 minutes wasperformed three times. Blocking One (Nacalai Tesque) was used andreacted at room temperature for one hour. Washing in PBS for 5 minuteswas performed three times. Reaction was performed for one hour with a400-fold diluted solution of the primary antibody polyclonal rabbitanti-human albumin antibody (DAKO) or a 300-fold diluted solution ofpolyclonal rabbit anti-human α-1-fetoprotein antibody (DAKO). Washing inPBS for 10 minutes was performed three times. Reaction was performed forone hour with 500-fold diluted solution of the secondary antibodyAlexaFluor[trademark] 466 goat anti-rabbit IgG antibody (MolecularProbes). Washing in PBS for 10 minutes was performed three times.Embedding was performed using Perma Fluor (Japan Tanner) and observationwas carried out with a microscope. The results are shown in FIG. 12 andFIG. 13. The presence of α-fetoprotein, and albumin was verified inhepatic lobule cell population. From this, hepatic lobule cellpopulation was found to produce actually these proteins.

Example 4 Incorporation of LDL by Hepatic-Lobule-Like Cell Cluster

1. Labeling of LDL with DiI

Human LDL (density: 1.019 to 1.063 g/ml) was isolated. The isolation wascarried out from a donor having normal lipoproteins by subjecting thesame [23] to ultracentrifugation sequentially, dialysis withsaline-EDTA, and then sterilization by filtration with a 0.2 μm filter.Next, the above LDL was incubated with1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI; MolecularProbes) (3 mg/ml) in 100 ml of DMSO at 37° C. for 8 hours in 0.5% bovineserum albumin (BSA)/PBS to label the lipoprotein (LDL) with DiI.Thereafter, this lipoprotein was dialyzed with PBS and filtered beforeuse.

2. Incorporation of LDL

In order to examine the incorporation of DiI-LDL, a hepatic lobule cellpopulation, which was differentiated from ADMPC, was incubated inserum-free DMEM containing 10 μg/ml DiI-LDL at 37° C. for 3 hours. Next,the cells were washed three times and mounted over Permaflur[24]. Thisslide was checked using a confocal laser scanning microscope (FloviewFV1000, Olympus). The results are shown in FIG. 14. It is clear fromFIG. 14 that DiI-LDL has been incorporated massively in the cytoplasmicregions of the hepatic lobule cell population. From this, it could beconfirmed that the hepatic-lobule-like cell cluster obtained by thepresent invention had a function for incorporating LDL.

Example 5 Accumulation of Glycogen by Hepatic-Lobule-Like Cell Cluster(PAS Staining)

A differentiated hepatic-lobule-like cell cluster was fixed with 4%paraformaldehyde and embedded in paraffin. The sample was sliced in athickness of 5 μm to prepare a section. Thereafter, this section wasoxidized with 1% periodic acid for 5 minutes and rinsed three times withdeionized water (dH₂O). Next, treatment with a Schiff reagent for 15minutes and rinsing for 5 to 10 minutes with dH₂O were performed.Furthermore, this section was counterstained for 1 minute with Mayer'shaematoxylin, rinsed with dH₂O and observed with a light microscope. Theresults are shown in FIG. 15. From FIG. 15, the presence of manyperiodic acid Schiff stain (PAS)-positive germ cells could be observedin the hepatic-lobule-like cell cluster. From this, thehepatic-lobule-like cell cluster obtained by the present invention wasshown to have a function for accumulating glycogen.

Example 6 Generation of Urea by Hepatic-Lobule-Like Cell Cluster 1.Generation of Urea

A hepatic-lobule-like cell cluster was incubated for two hours in 5 mlof Hank's balanced salt solution (Gibco) containing 5 mM NH₄Cl. The ureaconcentration in 0.5 ml of supernatant was measured using QuantiChromUrea Assay Kit (Bioassay Systems). The obtained concentration wasmultiplied by the total volume of supernatant to calculate the totalamount of urea generated. HepG2 was used as control.

2. Measurement of DNA Amount

Hank's balanced salt solution containing NH₄Cl was eliminated and thehepatic-lobule-like cell cluster was washed in PBS. A buffer solutionwas added, and ultrasonication was performed to homogenize the cells. To50 μl of cell homogenate, 1 ml of buffer solution was added, 50 μl ofcoloring solution was further added, and stirring was performed. Thefluorescence value of the obtained solution was measured at 356 nmexcitation and 458 nm emission to determined the DNA concentration. Tocalculate the overall DNA, the DNA concentration was multiplied by thevolume of buffer solution added.

3. Amount of Urea Generated

The obtained total amount of urea generated was divided by the overallDNA amount to calculate the overall amount of urea generated. Theresults are shown in FIG. 16. The hepatic-lobule-like cell cluster wasfound to generate a sufficient amount of urea compared to HepG2. Fromthis, it could be confirmed that the hepatic-lobule-like cell clusterobtained by the present invention had sufficient detoxification action.

Example 7 Grafting Effect of Hepatic-Lobule-Like Cell Cluster in MouseHepatitis Model 1. Preparation of Mouse Hepatitis Model

A mouse hepatitis model was prepared by intraperitoneal injection intoNOD-SCID mouse of carbon tetrachloride (CCl₄) at 300 μl/kg, twice weeklyand for 12 weeks.

2. Grafting of Hepatic-Lobule-Like Cell Cluster

A hepatic-lobule-like cell cluster was washed with Hank's balanced saltsolution, centrifuged and pelleted. The above mouse was anaesthetizedwith sevofluene[25]. A celiotomy [26] was performed by left paramedianincision, the left kidney was exposed, the renal capsule[27] separatedto create a pocket. The pelletized cell population was injected andgrafted inside the created pocket. The abdominal wall was closed in twolayers.

3. Analyses

On day 10 of grafting blood was collected from the mice to measure bloodconcentration of bilirubin. The results are shown in FIG. 17. The bloodconcentration of bilirubin was found to decreased in the group graftedwith the hepatic-lobule-like cell cluster compared to the group with nografting performed. In addition, the following histological analysis wasperformed using the above left kidney. The left kidney grafted with thehepatic-lobule-like cell cluster was taken out and fixed immediatelywith 10% formalin. Next, embedding with paraffin was performed,haematoxylin and eosin staining (FIG. 18) and PAS staining (FIG. 20)were performed by similar methods to above. On one hand, kidney graftedwith hepatic-lobule-like cell cluster was also used to performimmunofluorescence staining for albumin. First, the tissue was placed inOCT-compound (Sakura Fineteck Inc.) and frozen immediately. A 7 μmsection was prepared and fixed in 4% paraformaldehyde/PBS (WAKO) for 30minutes. This fixed section was incubated with a blocking solution(Blocking One; Nacalai Tesque). Next, incubation with anti-human ALBantibody (adsorbed with goat polyclonal, cow, mouse and pig ALB andaffinity-purified; Bethyl Laboratories), followed by AlexaFluor 546donkey anti-goat IgG antibody (Molecular Probes), was performed. Thetreated section was observed with a fluorescence microscope (BX61,Olympus) (FIG. 19). Grafting of hepatic-lobule-like cell cluster couldbe confirmed from FIG. 18. ALB antibody and PAS staining-positive germcells could be observed from each of FIG. 19 and FIG. 20, revealing thatalbumin was expressed and that glycogen has accumulated. These indicatethat the liver function is maintained. From the above, thehepatic-lobule-like cell cluster obtainable by the methods of thepresent invention was found to be effective in treating a disease thatoccurs due to a decrease in liver function.

Example 8 Collection of Adiopse Tissue from Human Subject

Excessive adipose tissue was extracted during gastric cancer operationfrom ten subjects (four males and six females) from whom informedconsents were received. The protocol was according to Osaka UniversityGraduate School of Medicine Review Boards for Human Research. Allsubjects were fasted for at least 10 hours. The age of the subjects was55±5 years (average±SE; 40 to 60 years range). There were no subjectbeing administered with a steroidal agent or TZD. From the subjects, 1to 10 g of abdominal subcutaneous (outside of fascial surface) adiposetissue and dorsal mesogastrium adipose tissue.

Isolation and Culture of ADMPC

The adipose tissue was sliced, and then digested in Hank's bufferedsaline solution (HBSS) containing 0.075% collagenase (Sigma ChemicalCo.) for one hour while shaking in a 37° C. water bath. The digestionproduct was filtered with Cell Strainer (BD Bioscience) and centrifugedat 800 g[9] for 10 minutes. Erythrocytes were eliminated by the densitymethod using Lymphoprep (d=1.077) (Nycomed), and the obtained preadiposetissue-derived multipotent progenitor cell population was cell-plated inDMEM containing 10% fetal bovine serum (Hyclone) [10]. Cells wereattached by culturing for 24 hours, washed and then treated with EDTA toobtain ADMPCs. Next, ADMPCs were plated in Culture Medium I: 60%DMEM-low glucose, 40% MCDB201, 10 μg/mL EGF, 1 nM dexamethasone, 100 μMascorbic acid, and 5% FBS, at a density of 10,000 cell/cm² over humanfibronectin coated dishes, subcultured 3 to 5 times, and used in theexperiments. The micrograph of ADMPC cultured for 10 days is shown inFIG. 21.

Example 9 ADMPC Expression Characteristics

Total RNA was isolated from ADMPC and adiposphere, using Mag-Extractorkit (TOYOBO) according to the protocols recommended by the manufacturer.Dnase treatment was carried out on 500 ng of total RNA and cDNA wassynthesized using Superscript III reverse transcriptase RNase H (−)(Invitrogen). RT-PCR was carried out for Islet-1, Nk×2.5, GATA-4, α-CA,MLC, MHC, nestin, neurofilament 68, somatostatin, snail, slug, andGAPDH, using KOD-plus (TOYOBO), under the following conditions: 40cycles of degeneration at 94° C. for 2 minutes, then degeneration at 94°C. for 15 seconds, annealing at predetermined temperature for 30 secondsand elongation at 68° C. for 30 seconds elongation. The annealingtemperature and primer sequence of each gene is shown in Table 2. As acontrol, adipose tissue-derived stem cell (hereinafter referred to as“ADSC”) obtained according to methods described in Japanese Translationof PCT Application No. 2005-502352 was used. The obtained amplificationproducts were electrophoresed on a 2% agarose gel. The results are shownin FIG. 22 to FIG. 24. Similarly to adipospheres, ADMPC was suggested toexpress snail and slug, which are markers of neural crest cells. Inaddition, ADMPC was shown to express a marker of absence ofdifferentiation, in particular Islet-1, known as a marker for cardiac,hepatic and pancreatic progenitor cells. ADSC did not express Islet-1,which confirms that the obtained ADMPC is a different cell from ADSC.

TABLE 2 Annealing temper- SEQ ature Gene Primer sequence ID (° C.)Islet-1 GTCAGTGGTGGACCTGACCT 1 60 AGGGGAGATTCAGTGTGGTG 2 Nkx2.5GGTGGAGCTGGAGAAGACAGA 3 60 CGACGCCGAAGTTCACGAAGT 4 GATA-4ACCAGCAGCAGCGAGGAGAT 5 60 GAGAGATGCAGTGTGCTCGT 6 α-CAGGAGTTATGGTGGGTATGGGTC 7 60 AGTGGTGACAAAGGAGTAGCCA 8 MLC-2vGCGCCAACTCCAACGTGTTCT 9 60 GTGATGATGTGCACCAGGTTC 10 MHCGGGGACAGTGGTAAAAGCAA 11 60 TCCCTGCGTTCCACTATCTT 12 GAPDHGTCAGTGGTGGACCTGACCT 13 60 AGGGGAGATTCAGTGTGGTG 14 nestinGGCGCACCTCAAGATGTCC 15 60 CTTGGGGTCCTGAAAGCTG 16 NeurofilamentATGAGTTCCTTCAGCTACGAGC 17 60 68 GGGCATCAACGATCCAGAGC 18 somatostatinGCTGCTGTCTGAACCCAAC 19 60 CGTTCTCGGGGTGCCATAG 20 snailAATCGGAAGCCTAACTACAGCG 21 60 GTCCCAGATGAGCATTGGCA 22 slugAAGCATTTCAACGCCTCCAAA 23 60 AGGATCTCTGGTTGTGGTATGAC 24

In addition, ADMPC was checked for the expression of Sca-1 and ABCG2,which are markers for the absence of differentiation, by as thusdescribed carrying out real time PCR with Applied Byosystems[11] 7900Fast Real-Time PCR system. The TaqMan probes used are shown in Table 3.The results are shown in FIGS. 25 and 26. ADMPC was confirmed to expressSca-1 and ABCG2.

TABLE 3 Gene name Reference sequence Assay ID Sca-1 NM_000332.2Hs00165656_m1 ABCG2 NM_004827.2 Hs00184979_m1

In order to check additional characteristics of ADMPC, ADMPCs isolatedfrom adipocytes were subjected to FACS. ADMPCs were separated from aculture dish with a 0.5 g/L-trypsin/0.53 mM-EDTA solution and suspendedin a Dulbecco's Phosphate-buffered Saline (DPBS, Nacalai Tesque)containing 0.1% FBS. A given quantity (5×10⁵ cells) was incubated withfluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodiesagainst human CD31 (BD PharMingen), CD105 (Ancell) and CD133 (R&D),phycoerythrin (PE)-conjugated mouse monoclonal antibodies against humanCD29, CD34, CD45, CD56, CD73, CD166 (BD PharMingen), CD44, or CD166(Ancell), at 4° C. for 30 minutes. In addition, the incubated cells wereincubated with mouse monoclonal antibodies against human SSEA-4,TRA-1-60, TRA-1-81 (Chemicon), ABCG-2, CD117 (BD PharMingen), orfibroblast/epithelial cell (AbD Serotec), as well as non-specific mouseantibody used as negative control, at 4° C. for 30 minutes. Afterwashing with DPBS, the cells were incubated with PE-labeled goatanti-mouse Ig antibody (BD PharMingen), at 4° C. for 30 minutes. Afterwashing three times, the cells were resuspended with DPBS, analysis wascarried out by flow cytometry using FACSCalibur flow cytometer andCellQuest Pro software (BD Biosciences). The results are shown in FIG.27 and FIG. 28. Compared to the control, the expression did almost notvary in ADMPC for the markers of hematopoietic system cell andhematopoietic stem cell (CD45, ABCG-2, CD34, CD133) (Mitchell et al.,Stem Cells, 24, 376-85 (2006)) and endothelial cell (CD31), surfaceantigen c-Kit (CD117), as well as surface markers for given ES cell andEG (embryonic germ) cell (TRA-1-60 and TRA-1-81) (James et al., 1998 andShamblott et al., Proc. Natl. Acad. Sci. USA 95, 1372613731 (1998)). Onthe other hand, from FIG. 27, it was observed that the expression waselevated for the surface markers of mesenchymal cell and/or nervous stemcell (CD29, CD44, CD73 and CD105) (Mitchell et al., Stem Cells, 24,376-85 (2006) and Barry et al., Biochem. Biophys. Res. Commun. 265,134139 (1999)) and the stage-specific embryonic antigen marker (SSEA-4)(Kannagi et al., EMBO J. 2, 23552361 (1983)). In addition, from FIG. 28,in contrast to 83.4% of cells positive for fibroblast surface antigen(Zuk et al., Mol. Biol. Cell. 13, 4279-4295 (2002)) in ADSC byconventional method, there are only 23% in ADMPC, demonstrating thatthere is little contamination by fibroblasts. From these, it was shownthat ADMPC according to the present invention has high differentiationcapability, and furthermore, the method of the present invention allowshigh purity ADMPC to be obtained.

Example 10 Verification of Multipotentiality of ADMPC

1. Differentiation Capability into Pancreatic Cell

ADMPCs were differentiated into pancreatic endocrine cells by themethods described in WO 2007/039986. The obtained pancreatic endocrinecells are shown in FIG. 29. The ADMPCs were verified to be capable ofdifferentiating into pancreatic endocrine cells, that is to say, to havefunctions as pancreatic progenitor cells. In addition, the efficiency ofdifferentiation from such ADMPCs into pancreatic endocrine cells washigher than that from ADSCs (data not shown).

2. Differentiation Capability into Hepatocyte

DMSO (0.1%), HGF (10 ng/mL), bFGF (10 ng/mL), and oncostatin M (10ng/mL), which are known to cause a hepatic progenitor cell todifferentiate into a hepatoblast/hepatocyte were added to a CultureMedium I to prepare Culture Medium II. ADMPCs were cultured for 14 daysin Culture Medium II to obtain hepatocytes. The obtained hepatocytes areshown in FIG. 30. The ADMPCs were verified to be capable ofdifferentiating into hepatocytes, that is to say, to have functions ashepatic progenitor cell. In addition, the efficiency of differentiationfrom the ADMPCs into hepatocytes was higher than that from ADSCs (datanot shown).

In addition, real time PCR was performed as thus described for theobtained hepatocytes with Applied Byosytems[12] 7900 Fast Real-Time PCRsystem to check the expression of α-fetoprotein (AFP), albumin, CYP1B1and glutamine synthase, which are markers that show differentiation intohepatocyte. ADMPCs were used as controls. The results are shown in FIGS.31 to 34. The expression of these genes was verified to be elevated inthe obtained hepatocytes.

3. Differentiation Capability into Cardiac MyocyteDifferentiation/Induction from ADMPC to Cardiac Myoblast

ADMPCs were cultured in Culture Medium I containing DMSO (Group 1) orOP9 culture supernatant (Group 2) for 14 days and the expression ofgenes in the obtained cells was checked by RT-PCR as described above. Ascontrol, ADMPCs cultured in Culture Medium I were used. The results areshown in FIG. 35. It could be verified that α-CA and MLC, which aremarkers of cardiac myoblast, were expressed in the cells from Groups 1and 2, that is to say, ADMPCs were differentiated/induced into cardiacmyoblasts.

Examination of Differentiation/Induction Period into Cardiac Myoblast

Next, ADMPCs were cultured in the presence of DMSO for 1, 2, 3, 4, 5, 7,10 and 14 days to check the differentiation into cardiac myoblasts. Ascontrols, ADMPC and cardiac myocyte were used. The results are shown inFIGS. 36 to 40. It was found that culturing in the presence DMSO allowedcardiac myoblasts to be obtained.

Preparation of Sheet Containing Cardiac Myoblasts

The obtained cardiac myoblast were cultured in Culture Medium Icontaining DMSO in a thermosensitive culture dish (CellSeed Inc.) at 37°C. to form a cell population. The cell population was detached byincubating at 20° C. or lower for 30 minutes to obtain a sheetcontaining cardiac myoblasts (refer to FIG. 41). The obtained sheet wasused in the following grafting experiment.

Grafting of Sheet to Myocardial Infarction Model Rat

A myocardial infarction model rat was prepared by ligating the coronaryartery of a nude rat. Then, an ADMPC-derived sheet containing cardiacmyoblasts was grafted to the injured region. Cardiac function wasevaluated at two weeks before grafting, before grafting, and two weeks,four weeks and 16 weeks after grafting by measuring the diameter atend-diastole (LVDd), the diameter at end-systole (LVDs), the leftventricular ejection fraction (% EF) and the left ventricular internaldiameter shortening fraction (% FS) with echo. As a control, a sheetcontaining ADMPCs formed as described above was used. The results areshown in FIGS. 42 to 49. Wall motion was observed in rats at 2 weeks and10 weeks after grafting the sheet containing cardiac myoblasts, showingthat cardiac function was remarkably improved. In contrast, in ratsgrafted with a sheet containing ADMPCs, although wall motion wasobserved after 2 weeks, it was not observed after 10 weeks. In addition,the LVDd started to extend and the EF decreased at week 8 and later withthe sheet containing cardiac myoblast; in contrast, LVDd and EF wereboth maintained with the sheet containing ADMPC-derived cardiac myoblast(ADMPC-derived cardiac myoblast is obtained by treating ADMPC in 0.1%DMSO for 48 hours), showing an improvement in cardiac function.

Histological Analysis of Heart Grafted with the Sheet

At 12 weeks and 16 weeks after grafting, rats were sacrificed and thehearts were extracted. The extracted hearts were fixed with a 4%paraformaldehyde solution, and then substituted with ethanol at 70%. Thefixed hearts were cut into a width of a few millimeters and solidifiedwith paraffin to prepare blocks. The obtained paraffin blocks werethin-sectioned to 2 μm using a microtome, pasted onto a slide glass anddried. The obtained thin sections were subjected to haematoxylin-eosinstaining and immunohistological staining as follows.

A. Haematoxylin-Eosin Staining

Thin sections were de-paraffinized and washed with water. Staining withhaematoxylin solution was performed for 10 minutes and coloration wasperformed for 3 minutes in lukewarm water. After rinsing, staining witheosin was performed for 5 minutes. Fractionation and dehydration wereperformed with alcohol. After clarifying with xylene, mounting wasperformed, and observation was performed with a microscope. The resultsare shown in FIGS. 50 and 51. It was verified that the grafted sheet hadengrafted.

B. Immunohistological Staining

Thin sections were de-paraffinized and washed with water.Immunostimulation treatment was performed, immersion into TBS-T addedwith 10% normal goat serum was performed, and blocking was performed at4° C. for 24 hours. After washing with TBS-T, primary antibody diluted100-fold with TBS-T added with 10% normal goat serum was added drop-wiseto the sample and reacted at 37° C. for one hour. As primary antibodies,anti-human a-cA antibody (American Research Products, Inc), anti-humanMHC antibody (Upstate cell signaling solutions) and anti-human HLA-ABCantibody (Hokudo Co., Ltd.) were used. After washing with TBS-T, SimpleStain Rat MAX-PG (Nichirei Bioscience Corp.) was added drop-wise andreacted at room temperature for 30 minutes. After washing with TBS-T,Simple Stain AEC Solution (Nichirei Bioscience Corp.) was addeddrop-wise and colored while examining under microscope. After washingwith water, nuclear staining was carried out by staining withhaematoxylin solution for 3 minutes. After washing with water, non-watersoluble mounting agent (Nichirei Bioscience Corp.) was added drop-wise,mounting was performed with a cover glass, and observation was carriedout with a microscope and a fluorescence microscope. In addition, inorder to evaluate the amount that differentiated into cardiac muscle andthe amount of remaining cardiac muscle in the region where the sheetscontaining ADMPC and ADMPC-derived cardiac myoblast were grafted, thethickness of α-CA antibody-positive region was measured. The results areshown in FIGS. 51 to 61. It could be verified that the site where thesheet containing cardiac myoblasts was grafted was HLA-ABC-positive,that is to say, human-derived, expressed α-CA and MHC, wasdifferentiated into cardiac muscle, and that the expression of α-CA wasalso maintained in the remaining cardiac muscle. Consequently, it wasfound that the grafted cardiac myoblasts transdifferentiated intocardiac myocytes and that the remaining cardiac muscle was protected.

4. Differentiation Capability into Adipocyte

ADMPCs were cultured using the adipocyte differentiation agent PPAR-yagonist to be differentiated into adipocytes. The obtained adipocyteswere oil red O-stained to measure the lipid content. ADSCs were used ascontrol. The results are shown in FIGS. 62 and 63. ADMPCs were found tobe capable of differentiating into adipocytes, that is to say, to havefunctions as adipose progenitor cells. In addition, the efficiency ofsuch differentiation was high compared to ADSCs. From the above it wasverified that ADMPCs were cells that have the capability ofdifferentiating into multipotent cells.

5. Differentiation Capability into Bone Tissue

ADMPCs were cultured for seven days in DMEM containing 10 nMdexamethasone, 50 mg/dl ascorbic acid 2-phosphate, 10 mMβ-glycerophosphate (Sigma), and 10% FBS to induce differentiation intobone tissue. The differentiation state was verified by alizarin redstaining and alkaline phosphatase (ALPase) activity. Regarding alizarinred staining, the obtained cells were washed three times and fixed withanhydrous ethanol. After fixation, the cells were stained with 1%alizarin red S in 0.1% NH₄OH (pH 6.5) for 5 minutes and washed with H₂O.Regarding ALPase activity, well known methods were followed (Bessey, O.A. et al., J. Biol Chem. 164, 321-329 (1946)). Describing in detail,cells were washed three times, then, homogenized at 0 to 4° C. in 1 mlof 0.9% NaCl and 0.2% Triton X-100 using a glass homogenizer, andcentrifuged at 1200 g for 15 minutes. The ALPase activity of thesupernatant solution was checked using p-nitrophenyl phosphoric acid(p-NP) as substrate. Specifically, this supernatant solution was assayedin a 0.5M Tris/HCl buffer solution (pH 9.0) containing 0.5 mM pNP and0.5 mM MgCl₂. The reaction mixture was incubated at 37° C. for 30minutes and the reaction was stopped by the addition of 0.25 volume of1N NaOH. The hydrolysis of pNP was monitored as the change in the valueof the absorbance at 410 nm of the spectrophotometer. p-nitrophenol wasused as a standard. One activity unit was defined as the amount thathydrolyzes 1 nmol of p-NP in 1 minute. The ALPase activity per cell wascalculated based on the DNA amount. The DNA content was measured by animprovement of a generic method (Labarca, C. et al., Biochem. 102,344-352 (1980)). After washing, the cells were homogenized at 0 to 4° C.in 1 ml of 2M NaCl/25 mM Tris-HCl (pH 7.4). After centrifugation at12000 g for 10 minutes, 25 ml of 5 mg/ml bis-benzimidazole was added to100 ml of supernatant solution. With a spectrofluorometer, excitationwas set to 356 nm, and the fluorescence spectrum at 458 nm lightemission was monitored. DNA concentration was checked using a standardcurve constructed from various concentrations of DNA from calf thymusgland. The results are shown in FIGS. 44 and 45. In comparison to ADSCusing conventional method, the ADMPCs according to the present inventionshowed strong positivity in alizarin red staining and AP activity,verifying that they were easily induced to differentiate into bonetissue. From the above, ADMPCs were shown to have the capabilities todifferentiate into tissues cells from multiple lineages including fromthe pancreas, the liver, the heart, fat, bone and the like.

INDUSTRIAL APPLICABILITY

According to the present invention, a method for obtaining a hepaticlobule cell population from an adipose-tissue-derived cell, a hepaticlobule cell population obtainable thereby, a method of screening for asubstance that promotes or inhibits formation of hepatic lobule and fora substance that causes the activity of a hepatic lobule to increase ordecrease, and a kit therefor, and the like can be obtained, all of whichare useful, for example, in the fields of preventing and treatingcirrhosis and hepatic cancer, and the fields of, for example, researchinto hepatic regeneration.

SEQ ID NO:1: Islet-1 forward primerSEQ ID NO:2: Islet-1 reverse primerSEQ ID NO:3: Nk×2.5 forward primerSEQ ID NO:4: Nk×2.5 reverse primerSEQ ID NO:5: GATA-4 forward primerSEQ ID NO:6: GATA-4 reverse primerSEQ ID NO:7: α-CA forward primerSEQ ID NO:8: α-CA reverse primerSEQ ID NO:9: MLC2v forward primerSEQ ID NO:10: MLC2v reverse primerSEQ ID NO:11: MHC forward primerSEQ ID NO:12: MHC reverse primerSEQ ID NO:13: GAPDH forward primerSEQ ID NO:14: GAPDH reverse primerSEQ ID NO:15: nestin forward primerSEQ ID NO:16: nestin reverse primerSEQ ID NO:17: Neurofilament 68 forward primerSEQ ID NO:18: Neurofilament 68 reverse primerSEQ ID NO:19: somatostatin forward primerSEQ ID NO:20: somatostatin reverse primerSEQ ID NO:21: snail forward primerSEQ ID NO:22: snail reverse primerSEQ ID NO:23: slug forward primerSEQ ID NO:24: slug reverse primer

1. A method for obtaining a hepatic-lobule-like cell cluster from anadipose-tissue-derived cell, the method comprising: culturing anadipose-tissue-derived cell.
 2. The method according to claim 1,comprising the step of obtaining a hepatic-lobule-like cell cluster froman undifferentiated cell.
 3. The method according to claim 1, comprisingthe steps of: (a) obtaining an undifferentiated cell from anadipose-tissue-derived cell; and (b) obtaining a hepatic-lobule-likecell cluster from the undifferentiated cell.
 4. The method according toclaim 2, wherein the step of obtaining a hepatic-lobule-like cellcluster from an undifferentiated cell is a step of producing ahepatic-lobule-like cell cluster by culturing an undifferentiated cellin a suspended state.
 5. A hepatic-lobule-like cell cluster obtainableby the method according to claim
 1. 6. A hepatocyte contained in thehepatic-lobule-like cell cluster according to claim
 5. 7. A medicinalcomposition for preventing or treating a disease that occurs due todeterioration in a liver function, the composition containing thehepatic-lobule-like cell cluster according to claim
 5. 8. Use of thehepatic-lobule-like cell cluster according to claim 5 for production ofa medicinal drug for preventing or treating a disease that occurs due todeterioration in a liver function.
 9. A method for treating orpreventing a disease that occurs due to deterioration in a liverfunction, said method comprising: administering the hepatic-lobule-likecell cluster according to claim
 5. 10. A method of screening for asubstance that promotes formation of hepatic lobule, said methodcomprising: adding a candidate substance to a culture medium whenculturing an adipose-tissue-derived cell to obtain a hepatic-lobule-likecell cluster, wherein the candidate substance is a substance thatpromotes formation of hepatic lobule when formation ofhepatic-lobule-like cell cluster is promoted in comparison withformation in a system not containing the candidate substance.
 11. Asubstance obtainable by the method according to claim 10, which promotesformation of hepatic lobule.
 12. A method of screening for a substancethat inhibits formation of hepatic lobule, said method comprising:adding a candidate substance to a culture medium when culturing anadipose-tissue-derived cell to obtain a hepatic-lobule-like cellcluster, wherein the candidate substance is a substance that inhibitsformation of hepatic lobule when formation of a hepatic-lobule-like cellcluster is inhibited in comparison with formation in a system notcontaining the candidate substance.
 13. A substance obtainable by themethod according to claim 12, which inhibits formation of hepaticlobule.
 14. A kit for screening for a substance that promotes orinhibits formation of hepatic lobule to be used in the method accordingto claim
 10. 15. A method of screening for a substance that causes anactivity of a hepatic lobule to increase, said method comprising:culturing, in a culture medium containing a candidate substance, ahepatic-lobule-like cell cluster that has been obtained by culturing anadipose-tissue-derived cell, wherein the candidate substance is asubstance that causes the activity of a hepatic lobule to increase whenan activity of the hepatic-lobule-like cell cluster has increased incomparison with an activity in a system not containing the candidatesubstance.
 16. A substance obtainable by the method according to claim15, which causes an activity of a hepatic lobule to increase.
 17. Amethod of screening for a substance that causes an activity of a hepaticlobule to decrease, said method comprising: culturing, in a culturemedium containing a candidate substance, a hepatic-lobule-like cellcluster that has been obtained by culturing an adipose-tissue-derivedcell, wherein the candidate substance is a substance that causes anactivity of a hepatic lobule to decrease when an activity of thehepatic-lobule-like cell cluster has decreased in comparison with anactivity in a system not containing the candidate substance.
 18. Asubstance obtainable by the method according to claim 17, which causesan activity of a hepatic lobule to decrease.
 19. A kit for screening fora substance which causes an activity of a hepatic lobule to increase ordecrease, said kit being used in the method according to claim 15.